1. Field of the Invention
The present invention relates to the synthesis of xanthan gum by Xanthomonas campestris and particularly to methods for increasing synthesis by modifying the natural organism.
b 2. Background of the Invention
A number of microorganisms produce extracellular polysaccharides, also known as exopolysaccharides or EPS. The exopolysaccharide known as xanthan is produced by the bacterium Xanthomonas campestris. The strain X. campestris pv campestris is a causal agent of black rot of cruellets.
Xanthan itself is useful as a specialty polymer for a growing number of commercial applications. The exploitation of xanthan as a commercial product results from a successful screening effort by the Northern Regional Research Center to find useful water-soluble polysaccharide products to replace existing gums from plant and algal sources. The NERL discovered X. campestris NRRL B1459, a strain which produces a polymer that exhibits three desirable properties: (1) high viscosity at low concentrations; (2) pseudoplasticity; and (3) insensitivity to a wide range of temperature, pH, and electrolyte concentrations. Because of its special theological properties, xanthan is used in food, cosmetics, pharmaceuticals, paper, paint, textiles, and adhesives and otherwise in the oil and gas industry.
In addition, the polymer is readily produced by fermentation from D-glucose. The synthesis of xanthan is believed to be similar to exopolysaccharide synthesis by other Gram-negative bacteria, such as species of Rhizobium, Pseudomonas, Klebsiella, and Escherichia. The synthetic pathway can be divided into three parts: (1) the uptake of simple sugars and their conversion to nucleotidal derivatives; (2) the assembly of pentasaccharide subunits attached to an isopentenyl pyrophosphate carrier; and (3) the polymerization of pentasaccharide repeat units and their secretion. By comparison to the more advanced molecular genetic understanding of colanic acid synthesis by E. coli or alginate synthesis by P. aeruginosa, little is known about the genes, enzymes, or mechanisms that control the synthesis of xanthan by X. campestris.
Xanthan gum is usually produced by fermentation of X. campestris with glucose or corn syrup as the major carbon source. Although it is also possible to convert the glucose and galactose in hydrolyzed cheese whey to xanthan gum, wild-type strains of X. campestris utilize lactose poorly, and the whey must first be hydrolyzed enzymatically with lactase of .beta.-galactosidase. There are some suggestions that the .beta.-galactosidase of X. campestris has a low affinity for lactose, thereby accounting for the poor utilization or unhydrolyzed lactose. Attempts have been made to generate a strain of X. campestris that can utilize lactose more efficiently. Exogenous lac genes have been transferred into X. campestris using transposon Tn951 which was in turn inserted within the mobilizable broad host range plasmid RP1. However, the plasmid, and therefore the lac genes, were not stable in the absence of a plasmid-selective antibiotic. Other investigators isolated a spontaneous derivative of X. campestris B1459 that could convert unhydrolyzed lactose in whey to xanthan gum. However, the nature of the mutation was not known, and the strain proved to be unstable for xanthan production, losing considerable productivity within forty generations under non-selective conditions.
Other genetic manipulations of X. campestris are also desirable. For example, undesirable enzymes are sometimes produced that contaminate the xanthan product, limiting the usefulness of xanthan gum to a narrower range of situations than would otherwise be possible.
Accordingly, an increased understanding or the genetic control of xanthan production by X. campestris would be useful for improving the productivity of X. campestris for xanthan synthesis.
3. Description of Relevant Literature
A recent publication on the topic of molecular cloning of genes involved in the production of xanthan in Bartere et al., Int. J. Biol. Macrotool. (1986) 8: 372-374. A study showing that a mutation, which blocks exopolysaccharlde synthesis and prevents modulation of peas by Rhizoblum leguminosarum, was corrected by cloned DNA from the phytopathogen Xanthomonas is described in Borthakur et al., Mol. Gen. Genet. (1986) 203:320-323. Production of xanthan using Xanthomonas campestris, properties of xanthan, and commercial applications of xanthan are described in Rogovin et al. J. Biochem. Microbiol Technol. Eng. (1961) 3:51-63, and Kennedy et al., 1984, "Production, properties, and applications of xanthan", pp. 319-371 in M. E. Bushell (ed.), Progress in Industrial Microbiology, vol. 19, Elsevier, Amsterdam.
A number of publications have occurred after the filing of U.S. application Ser. No. 038,302 on Apr. 14, 1987, now abandoned. These include Harding et al., J. Bacteriol. (1987) 169:2854-1286, which describes genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in X. campestris. European Patent Application EP 0 233 019 A2, filed Jan. 29, 1987, describes a recombinant DNA plasmid for xanthan gum synthesis. Thorne et al., J. Bacteriol. (1987 ) 169:3593-3600 , describes clustering of mutations blocking synthesis of xanthan gum by X. eampestris.